PROJECT SUMMARY In breast cancer patients, the migration of cancer cells away from the primary tumor and their subsequent metastasis to distant organs is the leading cause of mortality. Metastatic cells escape the primary tumor and enter the bloodstream by developing actin-rich membrane protrusions called invadopodia that degrade the extracellular matrix (ECM) to allow invasion of surrounding tissues. The assembly of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. Deregulation in Rho GTPase signaling has been associated with all stages of cancer progression, including proliferation, invasion and metastasis. However, little is known about how they are activated, the time course of their activation, or the identity of their upstream regulators and downstream effectors. Our long term goal is to characterize the mechanisms of regulation of Rho GTPases that contribute to cancer cell metastasis, in particular to cancer cell migration and invasion. Our preliminary results show that RhoG plays a key role in the regulation of invadopodia formation in breast cancer cells. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation. To characterize the RhoG signaling components involved in invadopodia formation, we have performed a proteomic analysis of the RhoG interactome and identified several potential RhoG binding partners. This application focuses on a subgroup of functionally related proteins, which includes potential upstream and downstream components involved in RhoG-mediated signaling. The objective of this application is to characterize these novel protein- protein interactions and their role during invadopodia formation and cell invasion. We will test our hypothesis by pursuing two specific aims: Aim 1. To validate the RhoG interaction partners identified by mass spectrometry. Using a proximity- based labeling assay we have identified a network of potential RhoG-interaction proteins. The goal of this aim is to validate the interactions identified by mass spectrometry. Aim 2. Characterization of the role of the identified proteins during invadopodia formation. We have uncovered a protein interaction network that may play a role in the regulation of RhoG-mediated invadopodia formation. The goal of this aim is to characterize the role of the different RhoG interaction partners on the regulation of RhoG activity, invadopodia formation, and cell invasion. It is expected that the knowledge generated from these studies will advance our understanding of how Rho GTPase pathways regulate critical steps during cancer progression. Future work will build up on this application and extend these studies to dissect the function of this protein network during cell invasion and metastasis in vivo.